A rapid enzymatic procedure for production of 1-beta-D-(3H)arabinofuranosyl cytosine 5'-triphosphate.

A rapid method of sequentially phosphorylating picomole quantities of [³H]-araC to [³H]araCTP is described (ara = 1-β-d-arabinofuranosyl). The procedure utilizes a system of phosphorylating enzymes isolated from rat spleen and requires a single incubation step. The [³H]araCTP product is isolated by ion-exchange chromatography and analyzed by PEI-cellulose thin-layer chromatography. At low concentrations of [³H]araC as much as 80% can be phosphorylated to the triphosphate, and the produet may be obtained in radiochemical purity greater than 97%.     

Nerve growth factor has both mitogenic and antimitogenic activity

The present study demonstrates that nerve growth factor (NGF) possesses both antimitogenic and mitogenic activities. To this end, we have employed clonal PC12 rat pheochromocytoma cells and two PC12 variant sublines, U2 and U7. When PC12 cells are exposed to NGF in culture media that are otherwise either permissive (15% serum) or restrictive (1% serum) for proliferation, neuronal differentiation occurs and mitosis ceases. Variant lines of PC12 cells have been selected that continue to proliferate in the presence of NGF in permissive medium but which nevertheless retain NGF receptors and certain NGF responses. In contrast to the parent PC12 cells, when such variants were exposed to NGF in growth-restrictive media, cell proliferation was markedly stimulated. The mitogenic activity of NGF was detectable at 0.1 ng/ml (4 pM) and was maximal at 3 ng/ml (100 pM). Possible contamination of the NGF preparation by epidermal growth factor (EGF) or mitogenic proteolytic enzymes was ruled out by the use of anti-EGF and diisopropylfluoro-phosphate, respectively. These findings show that NGF shares the capacity to stimulate cell division with a variety of other peptide hormones and suggest that the mitogenic activity of NGF could play a role in development of the peripheral nervous system as well as in promotion of in vivo growth of certain neural crest-derived neoplasms.     

Estimation of branched-chain α-keto acids in blood by gas chromatography

Plasma or whole blood is treated with o-phenylenediamine dihydrochloride in phosphoric acid under conditions found spectrophotometrically to give maximum yields of the quinoxalinols. The quinoxalinols are extracted and, after removing phosphoric acid, etc., silylated with bis-trimethylsilyltrifluoracetamide in acetonitrile. Other solvents caused instability of the trimethylsilyl(TMS)-quinoxalinols. Gas chromatography on a packed column of trifluoropropyl silicone gave good separation of the TMS-quinoxalinols from one another and from other substances derived from blood. Some representative values for normal arterial and venous human and canine plasma are reported.     

Trace metal levels in commercially prepared tissue culture media

Four trace elements, lead, copper, tin and zinc, in addition to certain electrolytes, were measured in 11 commercially prepared tissue culture media. Glass media bottles and plastic tissue culture dishes and flasks were treated with a HCl acid solution to determine the amounts of trace metals leached from their surfaces. Zinc, lead and copper were detected in all media. Tin was detected only in RPMI Medium 1640, fetal bovine serum, minimum essential medium and penicillin-streptomycin. It is possible that a major cause of variability in tissue culture experimental results may be due to effects on growth caused by fluctuation in trace element contamination from batch to batch. Variability in establishing primary cultures of corneal endothelial cells was traced to high lead levels in commercially prepared tissue culture media. A strong case is made for continued diligent efforts to expand analytical horizons and our definition of substances in culture media.     

Release of the "ammonia effect" on three catabolic enzymes by NADP-specific glutamate dehydrogenaseless mutations in Saccharomyces cerevisiae

Mutations which inactivate the NADP-glutamate dehydrogenase (anabolic GDHase) pleiotropically release the ammonia inhibition (NH₄⁺ effect) on a number of distinct catabolic activities. In addition to releasing inhibition on several permeability functions (1), these mutations suppress the NH₄⁺ effect on the synthesis of arginase, urea amidolyase and allantoinase. They do not affect the NH₄⁺ effect on the NAD-glutamate dehydrogenase.Two mechanisms of action of these mutations have to be considered, namely a modification of the process of $\text{induction}$ (such as removal of inducer exclusion) and a suppression of $\text{nitrogen catabolite repression}$.